In humans, there are two isoforms of clathrin protein, CHC17 and CHC22, named for their encoding chromosomes. The CHC17 isoform is well studied and forms the outer layer of clathrin-coated vesicles, which sort cargo during receptor-mediated endocytosis from the plasma membrane (PM), endosomal traffic, and organelle biogenesis from the trans-Golgi network (TGN). The gene encoding CHC22 is present in humans and other vertebrates but evolved into a pseudogene in mice, so humans, but not mice, express CHC22 clathrin. The Brodsky laboratory has extensively studied CHC17 clathrin, contributing to general knowledge about its biochemistry, regulation and cell biology. Capitalizing on our familiarity with CHC17, we investigated the relative function of CHC22 and discovered that CHC22 participates uniquely in retrograde transport from endosomes to the TGN and thereby targets the GLUT4 glucose transporter to the insulin-responsive GLUT4 storage compartment (GSC) in human muscle and fat cells. Formation of the GSC and its release of GLUT4 to the PM in response to insulin is the major pathway for insulin-regulated glucose clearance. Aspects of this pathway are defective in type 2 diabetes and we showed that CHC22 associates with abnormal GLUT4- containing structures in type 2 diabetic patients. Furthermore, transgenic expression of CHC22 in mice, which use CHC17 to form their GSC, altered murine GSC properties leading to diabetic symptoms. These data indicate that CHC22-mediated membrane traffic is needed for human glucose metabolism, defining differences between human and mouse glucose homeostasis. Having demonstrated the physiological importance of CHC22, the goal of this proposal is to define the molecular basis for CHC22 action and address three hypotheses about its role in human glucose metabolism. The experiments proposed involve protein biochemistry, electron microscopy, in vitro cell biology, and in silico genetic analysis. Aim 1 will define CHC22 morphology, properties of self-assembly, CHC22 subunits and binding proteins, and structure to address the hypothesis that CHC22 has unique biochemical features that distinguish the stability and dynamics of the human GSC from its murine counterpart. Aim 2 will establish the regulation of CHC22 intracellular recruitment to membranes in HeLa cells, human myoblasts and transfected 3T3-L1 cells to address the hypothesis that CHC22 influences GLUT4 transport in muscle and fat cells because it competes with CHC17 to function preferentially in a localized step of membrane traffic. Aim 3 characterizes functional and biochemical differences between existing allelic variants of CHC22 to address the hypothesis that variant allotypes of CHC22 could contribute to metabolic differences between individuals, and exacerbate or protect against diabetes. The proposed research will characterize the molecular mechanism of CHC22 function, its cellular regulation and its relationship to membrane traffic pathways that malfunction in diabetes.